Sensitizing cells to proton radiation

ABSTRACT

Materials and methods for enhancing the effectiveness of proton radiation therapy (e.g., high linear energy transfer (LET) proton radiation therapy) against tumor cells are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/145,787, filed on Sep. 28, 2018, which claims benefit of priorityfrom U.S. Provisional Application Ser. No. 62/565,970, filed on Sep. 29,2017.

TECHNICAL FIELD

This document relates to materials and methods for enhancing theeffectiveness of radiation therapy against tumor cells, and moreparticularly to materials and methods for enhancing the effectiveness ofhigh linear energy transfer (LET) proton radiation therapy against tumorcells.

BACKGROUND

Radiation therapy is an effective treatment modality to control manycancers. Mainstream radiotherapy devices use X-rays as the form ofionizing radiation to damage malignant tumors. Unfortunately, X-rayradiation can have detrimental effects on normal tissue, since theenergy of the photons that make up X-ray beams is deposited in bothnormal and tumor tissue along the path of the beam. Proton radiationtherapy, on the other hand, can be controlled such that the protonsrelease their cancer-fighting energy at a selected position within thebody. This is achieved by controlling the energy characteristics of theproton beam, since the probability that a proton is absorbed by matteras it travels through a medium is dependent on its energy and thedistance it travels. The point at which the highest energy releaseoccurs is called the “Bragg peak.” Clinicians can modulate the Braggpeak's size and distance to cause the most damage to targeted tumorcells. In addition, proton beams can be conformed to the shape and depthof a patient's tumor, thus minimizing absorption in healthy tissues andorgans.

SUMMARY

This document is based, at least in part, on the discovery thatcompositions containing genistein can be useful for sensitizing tumortissue to radiation therapy, and particularly to radiotherapy with highLET proton radiation. Genistein acts as a selective agonist of estrogenreceptor beta and has significant cell-signaling activities that affecthow cells respond to radiation damage, beyond simply affecting theincidence of damage itself. As described herein, compositions (e.g.,suspension formulations) that contain genistein (e.g., genisteinnanoparticles) as the active ingredient can be used as sensitizingagents that enhance the effectiveness of radiation (e.g., high LETproton radiation) against tumors.

In a first aspect, this document features a method for sensitizing tumorcells to high LET proton radiation. The method can include contactingthe tumor cells with a composition containing one or morepharmaceutically acceptable carriers and nanoparticulate genistein at aconcentration between about 250 mg/mL and about 500 mg/mL, andsubsequently contacting the tumor cells with the high LET protonradiation. The tumor cells can be lung cancer cells, prostate cancercells, head and neck cancer cells, pancreatic cancer cells,colon/colorectal cancer cells, bladder cancer cells, thyroid cancercells, breast cancer cells, liver cancer cells, ovarian cancer cells,endometrial cancer cells, cervical cancer cells, kidney cancer cells,brain cancer cells, or melanoma cells. In some cases, the tumor cellscan be non-small cell lung cancer (NSCLC) cells. The method can includecontacting the tumor cells with the genistein composition about 24 hoursprior to contacting the tumor cells with the high LET proton radiation.

In another aspect, this document features a method for treating a mammalidentified as having a solid tumor and slated to undergo treatment withhigh LET proton radiotherapy. The method can include administering tothe mammal a composition containing one or more pharmaceuticallyacceptable carriers and nanoparticulate genistein at a concentrationbetween about 250 mg/mL and about 500 mg/mL. The method can includeadministering the composition to the mammal at about 24 hours before theonset of the high LET proton radiotherapy. The method can includeadministering the composition to the mammal throughout the course of thehigh LET proton radiotherapy. The method can include administering thecomposition to the mammal after completion of the high LET protonradiotherapy. The method can include administering the composition tothe mammal at least once a day during the course of the high LET protonradiotherapy treatment. The method can include administering thecomposition to the mammal at least twice a day during the course of thehigh LET proton radiotherapy treatment. The tumor can be a lung tumor, aprostate tumor, a head and neck tumor, a pancreatic tumor, acolon/colorectal tumor, a bladder tumor, a thyroid tumor, a breasttumor, a liver tumor, an ovarian tumor, an endometrial tumor, a cervicaltumor, a kidney tumor, a brain tumor, or a melanoma. In some cases, thetumor can be a non-small cell lung cancer (NSCLC) tumor. The mammal canbe a human. The method can include administering the composition to themammal at a dose of about 250 mg/day to 500 mg/day, about 500 mg/day to1,000 mg/day, about 1,000 mg/day to about 5,000 mg/day, or about 5,000mg/day to about 10,000 mg/day. The tumor can contain cells having a KRASor p53 signature. The tumor can contain cells that express ERβ. The canfurther include contacting the tumor in the mammal with high LET protonradiation. Due to the administration of the composition, the tumor canbe effectively treated with a dose of radiotherapy that is at least 10%less than the dose of radiotherapy that would be administered to acorresponding tumor in a mammal not treated with the composition. Due tothe administration of the composition, the tumor can be more effectivelytreated with the high LET proton radiotherapy, as compared to theeffectiveness of the same dose of high LET proton radiotherapyadministered to a corresponding tumor in a mammal not treated with thecomposition. The nanoparticulate genistein composition can have aparticle size distribution characterized by a d(0.5) less than or equalto 0.3 μm. The one or more pharmaceutically acceptable carriers can forma suspension medium, and can include a water soluble polymer comprisinga polyvinylpyrrolidone. The one or more pharmaceutically acceptablecarriers can include a nonionic surfactant, a diluent, or a buffer. Thenonionic surfactant can be present in an amount ranging from about 0.01%to about 10% by weight (w/w). The amount of water soluble polymer can beabout 0.5% to about 15% (w/w). The composition can contain a diluent anda preservative. The composition can further contain a non-ionicsurfactant. The nanoparticulate genistein can be present in thecomposition at an amount ranging up to about 50% (w/w) (e.g., an amountof about 20% to about 35% (w/w). The method composition can have ananoparticulate genistein concentration of about 325 mg/mL. Thecomposition can have a pH of about 2 to about 12. The composition can beformulated as a tablet, a capsule, or a gelatin capsule. The method caninclude administering the composition orally (e.g., as an oralsuspension), intramuscularly, subcutaneously, or intravenously.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph plotting a representative Bragg peak.

FIGS. 2A and 2B are graphs plotting the survival of H460 human non-smallcell lung cancer (NSCLC) cells treated with or without a nanoparticulategenistein composition 24 hours before being exposed to the indicateddoses of X-Ray (FIG. 2A) or high LET proton (FIG. 2B) radiation.

FIGS. 3A and 3B are graphs plotting the survival of H1299 human NSCLCcells treated with or without a nanoparticulate genistein composition 24hours before being exposed to the indicated doses of X-Ray (FIG. 3A) orhigh LET proton (FIG. 3B) radiation.

FIGS. 4A and 4B are graphs plotting the survival of MRC5 normal humanlung fibroblasts treated with or without a nanoparticulate genisteincomposition 24 hours before being exposed to the indicated doses ofX-Ray (FIG. 4A) or high LET proton (FIG. 4B) radiation.

DETAILED DESCRIPTION

Proton radiation differs from photonic gamma/X-ray radiation from aphysical perspective. Protons deposit their energy at a specific depthas they pass through various media, according to the energy of theproton. This aspect of proton radiation can be exploited inradiotherapy, by modulating the energy of the beam to target a depth oftissue where the maximal energy will be deposited. Thus, as protonradiation enters the body, less of its energy is deposited per unit pathlength (low unrestricted linear energy transfer or low LET protons) asit travels to the target area. At the end of its path (the Bragg peak),the amount of energy deposited per unit path length (unrestricted LET)is much higher (high LET protons). A graph showing a representativeBragg peak is shown in FIG. 1. To facilitate treating larger areas withprotons, the energy of the proton beam can be modulated to create aspread out Bragg peak (SOBP), such that the LET is lowest at theentrance and highest at the end of the SOBP.

In the experiments described herein, a R10.5M5 double scattered protonbeam (a proton beam with a maximum range of 10.5 cm where the SOBPcomprises the final 5 cm) was utilized with precise positioning ofculture dishes at specific points at the entrance, middle, and end ofthe SOBP. For this beam, the “low LET” was at 2 cm depth, correspondingto about 4 keV/μM, and the high LET was at 10 cm depth, corresponding toabout 30 keV/μM. As used herein, the term “low LET” refers to about oneto about ten (e.g., about one to three, two to four, three to five, fourto six, five to seven, six to eight, seven to nine, or eight to ten)keV/μM unrestricted LET as determined from tables of proton stoppingpower in liquid water, available from the National Institute ofStandards and Technology (stopping power and range tables for protons;“PSTAR”). The term “high-LET” refers to about 20 to 40 (e.g., 20 to 25,25 to 30, 30 to 35, or 35 to 40) keV/μM, again as determined from PSTARunrestricted LET tables.

The results discussed in the Examples below are consistent withLET-dependent radiosensitization by nanoparticulate genistein, where thehigher the LET that is delivered, the more the genistein is capable ofradiosensitizing the tumor cells. These effects also may be related tothe genetics of the tumor type.

As described herein, compositions containing genistein can be used asagents to increase the effectiveness of radiation (e.g., protonradiation, including high LET proton radiation) against tumor cells,either in vitro or in vivo. The methods provided herein thereforeinclude using genistein-containing compositions to sensitize tumor cellsto proton radiation therapy, and may result in prolonged survival (e.g.,progression-free or overall survival) of cancer patients treated asdescribed herein.

Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-chromen-4-one (IUPAC),5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one,5,7,4′-trihydroxyisoflavone, 4′,5,7-trihydroxyisoflavone) is aphytoestrogen in the category of isoflavones. Its chemical structure isshown in Formula (1):

Genistein is one of several known isoflavones that are normally found inplants. The main sources of natural genistein are soybeans and otherlegumes. Genistein also is commercially available, and may be obtainedin synthetic, purified form (e.g., from DSM Nutritional Products, Inc.,Parsippany, N.J.).

Genistein has antioxidant and anti-inflammatory properties (Verdrengh etal., Inflammation Res. 52(8):341-346, 2003; Polkowski and Mazurek, ActaPoloniae Pharmaceutica—Drug Research 57(2):135-155, 2000; and Kruk etal., Luminescence: J. Biol. Chem. Luminescence 20(2):81-89, 2005).Genistein also has effects on cell cycle division (Zhang, et al., Int. JOncol. 43(1):289-296, 2013) that likely are mediated by its selectiveactivation of estrogen receptor beta (ERβ) (Kuiper et al., Endocrinol139(10):4252-4263, 1998). In addition, genistein can inhibit proteintyrosine kinase activity, modulating signal transduction pathwaysinvolved in cell death and survival. The antioxidant properties ofgenistein may relate to genistein's ability to scavenge ROS, which aredirectly implicated in the formation of DNA double strand breaks. Thus,genistein may have beneficial effects in individuals exposed toradiation, acting to protect healthy tissue and mitigate the effects ofradiation in individuals accidently or intentionally exposed toradiation. As described herein, however, genistein also can act tosensitize tumor tissue to radiation, including high LET protonradiation.

As described elsewhere (see, e.g., U.S. Pat. No. 8,551,530), genisteinnanoparticles can have improved oral and/or parenteral bioavailabilityas compared to genistein that is not in nanoparticle form. Nanoparticleformulations can contain sub-micron size genistein particles, which canbe manufactured using a wet nanomilling process that reduces genisteinto a median particle size of less than 0.2 μm. See, e.g., U.S. Pat. No.8,551,530. In addition, pharmacokinetic experiments using such agenistein nanosuspension in mice, canines, and humans have demonstratedincreased oral bioavailability as compared to formulations containingnon-micronized genistein. See, e.g., FIGS. 4-7 of U.S. Pat. No.8,551,530.

In some embodiments, the genistein compositions used in the methodsprovided herein can be formulations that include genistein in a solutioncontaining one or more pharmaceutically acceptable carriers, excipients,and/or diluents. In some embodiments, the genistein compositions used inthe methods provided herein can be suspension formulations that includenanoparticulate genistein suspended in a medium containing one or morepharmaceutically acceptable carriers, excipients, and/or diluents.

Pharmaceutically acceptable carriers, excipients, and diluents suitablefor therapeutic use include those described, for example, in Remington'sPharmaceutical Sciences, Maack Publishing Co. (A. R. Gennaro (ed.),1985). In some cases, polyethylene glycol (PEG) can be used as a carrierin a composition that also contains genistein that is not innanoparticle form.

A genistein composition also can contain one or more other components,as described herein (e.g., one or more pharmaceutically acceptableexcipients that form a suspension medium, such as a water solublepolymer, a nonionic surfactant, a diluent, or a buffer). In someembodiments, a genistein composition can include a suspension ofgenistein (e.g., nanoparticulate genistein) in a non-aqueous medium,such as an edible lipid, oil, or fat from a plant or animal source(e.g., olive oil, sunflower oil, corn oil, soy oil, marine oil, coconutoil, palm oil, palm kernel oil, cotton seed oil, safflower oil, sesameoil, peanut oil, almond oil, cashew oil, pecan oil, pine nut oil,macadamia oil, orange oil, flax seed oil, lemon oil, walnut oil, borageoils, fish oils, and dairy derived fats). See, e.g., U.S. Pat. No.9,084,726. In some cases, a genistein composition can include asuspension (e.g., a suspension of nanoparticulate genistein) in a mediumincluding one or more water soluble polymers and one or more nonionicsurfactants. See, e.g., U.S. Pat. No. 8,551,530. Nonionic surfactantscan facilitate wetting and aid in preventing agglomeration ofnanoparticulate genistein, for example. Suitable nonionic surfactantsinclude, without limitation, polysorbates, poloxamers, polyoxyethylenecastor oil derivatives, bile salts, lecithin, 12-hydroxystearicacid-polyethylene glycol copolymer, and the like. In some embodiments, agenistein composition can include a non-ionic surfactant selected fromthe group consisting of polysorbate 80 (TWEEN© 80), polysorbate 20(TWEEN© 20), Poloxamer 188, and combinations thereof. The total nonionicsurfactant content in the genistein compositions utilized in the methodsprovided herein can range from about 0.01% to about 10% by weight (w/w)(e.g., about 0.2% to about 5% (w/w), about 0.2% to about 2% (w/w), about0.2% to about 1% (w/w), about 0.2% to about 0.6% (w/w), and about 0.2%to about 0.8% (w/w).

Water soluble polymers can serve to enhance the viscosity of asuspension and/or to stabilize nanoparticulate genistein againstparticle agglomeration or potential deleterious effects from otherformulation components, for example. Water soluble polymers arepharmaceutically acceptable polymers that can be dissolved or dispersedin water. Suitable water soluble polymers include, without limitation,vegetable gums (e.g., alginates, pectin, guar gum, and xanthan gum),modified starches, polyvinylpyrrolidone (PVP), hypromellose (IPMC),methylcellulose, and other cellulose derivatives (e.g., sodiumcarboxymethylcellulose, hydroxypropylcellulose, and the like). In someembodiments, the genistein compositions described herein can include apoloxamer (e.g., Poloxamer 188) as a water soluble polymer. Poloxamer188 is both a polymer and surfactant. The total water soluble polymercontent in a genistein composition for use in the methods providedherein can range from about 0.5% to about 15% (w/w) [e.g., about 1% toabout 10% (w/w), about 10% to about 15% (w/w), about 12% to about 15%(w/w), about 1% to about 8% (w/w), and about 1% to about 5% (w/w)].

Carriers suitable for use in the genistein formulations described hereinalso include pharmaceutically acceptable aqueous carriers such as,sterile water, physiologically buffered saline, Hank's solution, andRinger's solution. The formulations also can contain one or more buffers[e.g., one or more citrate buffers, phosphate buffers,tris(hydroxymethyl)aminomethane (TRIS) buffers, and/or borate buffers],to achieve a desired pH and osmolality. Injectable pharmaceuticalformulations typically have a pH in the range of about 2 to about 12. Insome embodiments, the genistein formulations used in the methodsprovided herein can have a pH that falls in a range that more closelyapproximates physiologic pH (e.g., about 4 to about 8, or about 5 toabout 7).

In some cases, the genistein compositions useful in the methods providedherein also can include one or more diluents. Suitable diluents includethose selected from, without limitation, pharmaceutically acceptablebuffers, solvents, and surfactants.

Moreover, in some embodiments, a genistein composition can include PVP(e.g., 5% PVP-K17) and polysorbate 80 (e.g., 0.2% polysorbate 80), aswell as phosphate buffered saline (PBS, e.g., 50 nM PBS). In some cases,an oral formulation of a genistein composition can contain PVP (e.g.,PVP-K25), polysorbate 80 (TWEEN® 80), and one or more preservatives(e.g., methyl paraben and/or propyl paraben). In addition, a compositioncan include a diluent such as a sodium chloride solution. In some cases,the particle size distribution of a nanoparticulate genisteincomposition can be d(0.5)≤0.5 microns (e.g., d(0.5)≤0.4 microns,d(0.5)≤0.3 microns, or d(0.5)≤0.2 microns). See, e.g., U.S. Pat. No.8,551,530. It is to be noted that while such genistein formulations arecharacterized as suspensions, depending on the carriers, excipients, anddiluents included in the suspension medium, a measurable amount ofgenistein also may be dissolved in the suspension medium.

A composition can contain genistein (e.g., nanoparticulate genistein orgenistein that is not in nanoparticle form) at a concentration betweenabout 100 mg/mL and about 500 mg/mL (e.g., about 100 mg/mL to about 400mg/mL, about 150 mg/mL to about 350 mg/mL, about 200 mg/mL to about 400mg/mL, about 250 mg/mL to about 350 mg/mL, about 250 mg/mL to about 500mg/mL, about 275 mg/mL to about 325 mg/mL, about 300 mg/mL to about 450mg/mL, or about 350 mg/mL to about 500 mg/mL). For example, a suspensionof nanoparticulate genistein can incorporate genistein in an amountranging from about 100 mg/mL to about 500 mg/mL (e.g., ranges from about100 mg/mL to about 400 mg/mL, about 150 mg/mL to about 350 mg/mL, about200 mg/mL to about 400 mg/mL, about 250 mg/mL to about 350 mg/mL, about275 mg/mL to about 325 mg/mL, about 300 mg/mL to about 450 mg/mL, orabout 350 mg/mL to about 500 mg/mL, or amounts of about 100 mg/mL, about150 mg/mL, about 200 mg/mL, about 250 mg/mL, about 275 mg/mL, about 300mg/mL, about 325 mg/mL, about 350 mg/mL, about 375 mg/mL, about 400mg/mL, about 450 mg/mL, or about 500 mg/mL).

The relative amount of genistein included in a composition can be variedto yield a formulation having a desired total content of genistein. Forexample, a composition (e.g., a suspension formulation as describedherein can include up to about 50% (w/w) genistein [e.g., about 50%(w/w), about 45% (w/w), about 40% (w/w), about 35% (w/w), about 30%(w/w), about 25% (w/w), about 20% (w/w), about 15% (w/w), about 10%(w/w), about 40% to about 50% (w/w), about 35% to about 45%, about 30%to about 40% (w/w), about 25% to about 35% (w/w), about 20% to about 30%(w/w), about 20% to about 35% (w/w), about 15% to about 35%, about 10%to about 30%, or about 10% to about 25%]. In some embodiments,nanoparticle genistein suspensions can provide increased bioavailabilityof genistein as compared to the bioavailability of genistein provided bysolution formulations (e.g., solutions containing a pharmaceuticallyacceptable PEG solvent or containing larger sized genistein material).As described in U.S. Pat. No. 8,551,530, for example, the combination ofhigh genistein loading and significantly increased bioavailability canprovide advantages, such as facilitating administration oftherapeutically effective amounts of genistein using much lower amountsof formulated drug substance, for example.

Genistein compositions can be formulated for administration by anysuitable method, depending upon whether local or systemic treatment isdesired and upon the area to be treated. For example, a genisteincomposition can be formulated for oral administration, parenteraladministration (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous drip),pulmonary administration (e.g., by inhalation or insufflation of powdersor aerosols or a nebulized mist), or by a combination of routes such asoral and parenteral administration. Administration can be rapid (e.g.,by injection) or can occur over a period of time (e.g., by slow infusionor administration of slow release formulations, such as fromsubcutaneous drug depots, slow short term intravenous injections, orslow release oral formulations).

Compositions and formulations for parenteral administration include, forexample, sterile solutions (e.g., sterile aqueous solutions orsuspensions) that also can contain buffers, diluents, and/or othersuitable additives (e.g., penetration enhancers, carrier compounds andother pharmaceutically acceptable carriers). Compositions formulated forparenteral delivery can be manufactured according to standard methods toprovide sterile compositions deliverable via, for example, intravenousinjection or infusion, intravascular injection, subcutaneous injection,or intramuscular injection. A genistein formulation (e.g., a suspensionof nanoparticulate genistein) can be prepared to have a viscositysuitable for the desired route of parenteral administration, and can bemanufactured and packaged in any manner suited to the desiredapplication, including, without limitation, as a formulation deliverablevia intravenous injection or infusion, intravascular injection,subcutaneous injection, or intramuscular injection. In some embodiments,a formulation as described herein can be contained in one or moreprefilled syringes or auto-injectors prepared for administration of agiven dose or range of doses of genistein.

Genistein compositions also can be formulated for oral administration.Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media (e.g., suspensions of genistein nanoparticles inedible oil), capsules, gel caps, sachets, and tablets. In someembodiments, a genistein composition can be prepared as a liquidsuspension that can be metered to deliver a desired dose, or can beincorporated into capsules (e.g., gelatin or soft capsules) suitable fordelivery of liquid formulations. Alternatively, formulations for oraladministration can be loaded into prefilled sachets or premetered dosingcups. In some embodiments, such genistein formulations also can includeone or more pharmaceutically acceptable sweetening agents,preservatives, dyestuffs, flavorings, or any combination thereof. Insome cases, genistein can be spray dried into a powder that subsequentlycan be hydrated to reconstitute a suspension.

Genistein compositions useful in the methods described herein canfurther include any pharmaceutically acceptable genistein salts, esters,or salts of such esters, or any other genistein compound which, uponadministration to an animal such as a human, is capable of providing(directly or indirectly) biologically active genistein or an activemetabolite or residue thereof. Accordingly, pharmaceutically acceptablesalts of genistein, prodrugs and pharmaceutically acceptable salts ofsuch prodrugs, and other bioequivalents can be used in the genisteincompositions described herein. The term “prodrug” indicates atherapeutic agent that is prepared in an inactive form and is convertedto an active form (i.e., drug) within the body or cells thereof by theaction of endogenous enzymes or other chemicals and/or conditions. Theterm “pharmaceutically acceptable salts” refers to physiologically andpharmaceutically acceptable salts of genistein (e.g., salts that retainthe desired biological activity of genistein without imparting undesiredtoxicological effects). Examples of pharmaceutically acceptable saltsinclude, for example, salts formed with cations (e.g., sodium,potassium, calcium, or polyamines such as spermine), acid addition saltsformed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, or nitric acid), and salts formed withorganic acids (e.g., glucuronic acid, acetic acid, citric acid, oxalicacid, palmitic acid, or fumaric acid). Depending on the route ofadministration, for example, genistein may be sulfated or in glucuronicacid form.

Compositions also can include other adjunct components conventionallyfound in pharmaceutical compositions. Thus, the compositions also caninclude compatible, pharmaceutically active materials such as, forexample, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or additional materials useful in physicallyformulating various dosage forms of the compositions provided herein,such as dyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents and stabilizers. Furthermore, the composition can bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings, and aromatic substances. Whenadded, however, such materials should not unduly interfere with thebiological activities of the genistein within the composition. Theformulations can be sterilized if desired.

This document provides methods for using genistein compositions such asthose described above. For example, the methods disclosed herein caninclude contacting a tumor cell, either in vitro or in vivo, with agenistein composition, such that the sensitivity of the tumor cell toradiation (e.g., higher LET proton radiation) is increased relative to acorresponding tumor cell that was not treated with the genisteincomposition.

In some embodiments, therefore, this document provides methods forsensitizing tumor cells to proton radiation (e.g., high LET protonradiation), where the methods include contacting the tumor cells with acomposition that contains a pharmaceutically acceptable carrier andgenistein (e.g., nanoparticulate genistein, where the compositioncontains nanoparticulate genistein at a concentration between about 250mg/mL and about 500 mg/mL). Various types of tumors/tumor cells can besensitized to radiation using the methods described herein. Theseinclude cells from solid tumors such as, without limitation, lung cancer(e.g., NSCLC) cells, prostate cancer cells, head and neck cancer cells,pancreatic cancer cells, colon/colorectal cancer cells, bladder cancercells, thyroid cancer cells, breast cancer cells, liver cancer cells,ovarian cancer cells, endometrial cancer cells, cervical cancer cells,kidney cancer cells, brain cancer cells, and melanoma cells, as well asnon-solid tumor cells such as non-Hodgkin lymphoma cells.

Tumor cells can be treated with a genistein composition prior to theirirradiation with, e.g., high LET proton radiation. For example, tumorcells can be contacted with a genistein composition about 7 days toabout 1 hour before radiation exposure. In some embodiments, the cellscan be contacted with a genistein-containing composition about 6 to 7days, about 5 to 6 days, about 4 to 5 days, about 3 to 4 days, about 60to 72 hours, about 48 to 60 hours, about 36 to 48 hours, about 24 to 36hours, about 18 to 24 hours, about 12 to 18 hours, about 10 to 12 hours,about 8 to 10 hours, about 6 to 8 hours, about 4 to 6 hours, about 2 to4 hours, or about 1 to 2 hours before irradiation. In some cases, tumorcells can be contacted with a genistein composition about 20 to 28 hours(e.g., about 22 to 26 hours, or about 24 hours) before exposure toproton radiation.

This document also provides methods for treating a mammal (e.g., ahuman, or a non-human mammal such as, without limitation, a non-humanprimate, dog, cat, rat, rabbit, pig, sheep, mouse, cow, or horse)identified as having a solid tumor, where the mammal will undergo protonradiotherapy treatment, such as treatment with high LET protonradiation. The methods can include administering to the mammal acomposition that contains a pharmaceutically acceptable carrier andgenistein (e.g., nanoparticulate genistein as described herein). Again,a number of different tumor types can be treated using the methodsprovided herein, including lung tumors (e.g., NSCLC tumors), prostatetumors, head and neck tumors, pancreatic tumors, colon/colorectaltumors, bladder tumors, thyroid tumors, breast tumors, liver tumors,ovarian tumors, endometrial tumors, cervical tumors, kidney tumors,brain tumors, and melanomas, as well as non-Hodgkin lymphoma. In someembodiments, the tumor can include cells having a particular geneticsignature or mutation (e.g., a KRAS or p53 signature or mutation; see,e.g., Loboda et al., BMC Medical Genomics 2010, 3:26; and Saleemuddin etal., Gynecol Oncol. 2008, 111(2):226-232) or the tumor can include cellsexpressing ERβ. Moreover, the mammal to be treated can be identified ashaving an alteration in one or more particular genes. For example, themammal may be identified as having a rearrangement of the ALK gene, theRET gene, or the ROS1 gene, a mutation in the AKT1 gene, the BRAF gene,the DDR2 gene, the EGFR gene, the HER2 gene, the KRAS gene, the MEK1gene, the NRAS gene, the PIK3CA gene, or the PTEN gene, and/or anamplification of the FGFR1 gene or the MET gene. Further, a mutation inthe androgen receptor (AR) gene or the HOXB13 gene may be causative of apatient's cancer and differentiate genistein effectiveness. Thedevelopment of cancer also may be linked to certain viral infections(e.g., human papillomavirus (HPV)). Viral-driven cancer maydifferentiate a tumor's response to radiation and/or genistein. Inaddition, inactivating mutations in DNA repair genes, such as ATM, ATR,BRCA1, BRCA2, PALB2, or MSH1, may affect how genistein modulates apatient's tumor-response to radiation. Methods for determining whether amammal includes a particular genetic signature, marker, or mutation aretypically well understood, and are described elsewhere.

The genistein-containing composition can be administered to the mammalbeginning about an hour to about a week (e.g., six to seven days, fiveto six days, four to five days, three to four days, two to three days,one to two days, 12 to 24 hours, six to 18 hours, six to 12 hours, threeto six hours, one to three hours, about seven, six, five, four, three,two, or one day, or about 18, 12, six, three, or one hour) before theonset of radiotherapy. Moreover, administration of thegenistein-containing composition can be continued throughout the courseof radiotherapy, and in some cases, after completion of the radiotherapy(e.g., for about one to seven days, one to two weeks, two to four weeks,four to six weeks, six to eight weeks, eight to ten weeks, or ten totwelve weeks). When administered, the genistein-containing compositioncan be given to the mammal at least once a day (e.g., once, twice, threetimes, or more than three times a day) before, during, and/or after thecourse of the radiotherapy treatment. In some cases, the administeringcan be such that the mammal receives a dose of about 100 mg/day to about10,000 mg/day (e.g., about 250 to 500 mg/day, about 500 to 1,000 mg/day,about 1,000 to about 5,000 mg/day, or about 5,000 to about 10,000mg/day).

The methods provided herein can include administering an effective doseof genistein to a mammal, where an “effective dose” provides aradio-sensitizing effect to cells of a particular type (e.g., tumorcells), enhancing a reduction in tumor size and/or number of tumor cellswhen combined with high LET proton radiation. The sensitization of atumor to radiation therapy by genistein administration can mean thatless radiation is needed for effective treatment than if genistein hadnot been administered. Thus, by use of the methods provided herein, atumor can be effectively treated with a dose of radiotherapy that is atleast about 5% (e.g., about 5%, about 10%, about 20%, about 30%, about50%, or at least 75%) less than the dose of radiotherapy that would haveto be administered to a corresponding tumor in a mammal not treated withthe genistein-containing composition. The sensitization of a tumor toradiation therapy by genistein administration also can mean that thesame dose of radiation has a greater therapeutic effect than it wouldhave if genistein was not administered. Thus, by use of the methodsprovided herein, a dose of radiotherapy can have a therapeutic effect(e.g., on the size of a tumor or the number of cancer cells) that is atleast about 5% (e.g., about 5%, about 10%, about 20%, about 30%, about50%, or at least 75%) greater than the same dose's effect on acorresponding tumor in a mammal not treated with thegenistein-containing composition.

The administering can be accomplished via any suitable route. In someembodiments, for example, a genistein composition containing a solutionof genistein or a suspension of genistein nanoparticles can beadministered orally (e.g., as a suspension) or parenterally (e.g., byinjection, such as subcutaneous, intravenous, or intramuscularinjection).

In some embodiments, the methods provided herein can include monitoringa mammal treated with genistein and proton radiation to determinewhether the number of cancer cells or the size of a tumor is diminishedafter treatment. Any appropriate method can be used to determine whetheror not the number of cancer cells or the size of a tumor present withina mammal is reduced. For example, imaging techniques can be used toassess the number of cancer cells and/or the size of a tumor presentwithin a mammal.

The invention will be further described in the following example, whichdoes not limit the scope of the invention described in the claims.

Example Effect of Genistein Pre-Treatment on Cell Survival afterExposure to Various Forms of Radiation

Studies were conducted to determine whether a nanoparticulate genisteinformulation affects cellular radiosensitivity to protons with increasingLET. Normal and cancer cell lines were exposed to low, intermediate, orhigh LET proton radiation. These experiments used the H1299 and H460NSCLC cell lines, which are relevant to lung cancer research duringmedical radiation exposure as well as nonmedical or accidental radiationexposure. These cell lines were derived from human cancer patients andhave well-known radiobiologic properties, with H1299 being relativelyless radiosensitive. Both lines harbor Ras pathway activating mutations(H460, KRAS; H1299, NRAS). MRC5 human lung fibroblasts also were used asrepresentative normal cells. Each cell line was obtained from theAmerican Type Culture Collection (ATCC) and tested for Mycoplasmaregularly. Clonogenic cell survival studies were performed as describedelsewhere (Cengel et al., Neoplasia 9(4):341-348, 2007). Briefly, cellswere plated 24 hours prior to radiation at about 50% confluence. Afterallowing attachment, culture media was supplemented with 5 μMnanoparticulate genistein or the equivalent volume of inactive vehicle.Cells were radiated using either 250 KVp x-rays or protons with lowervs. higher LET. To vary the proton LET, cell dishes were placed at adepth of 2 cm (D2, low LET) or 10 cm (D10, high LET) in a R10.5M5 doublescattered proton beam—a double scattered proton beam with a maximumrange of 10.5 cm and a modulated depth of 5 cm, meaning that theSpread-out Bragg peak (SOBP) began roughly at a depth of 5.5 cm. In thisconfiguration, the D2 and D10 positions corresponded to the entrance andterminal portions of the SOBP, respectively. After exposure toradiation, cells were trypsinized into a single cell suspension andre-plated at various densities without nanoparticulate genistein. Abouttwo weeks later (typically 13 to 16 days, depending on colony size),plates were rinsed and stained using crystal violet/ethanol, andcolonies were enumerated using an Oxford Optronix automated colonycounter with a minimum threshold size of 50 cells to define a colony.All experiments were performed a minimum of three times with at leastsix replicate dishes per condition. Data were then fit using alinear-quadratic (LQ) function of the form:

Surviving Fraction (Dose)=e ^(−(αD+βD) ² ⁾

Relative radiosensitivity was numerically compared using a standardvariable, Do (the dose of radiation needed to reduce the survivingfraction by a factor of 1/e), which was determined from the positivesolution to the quadratic equation of the form:

βD ² +αD−1=0,

where α and β are determined from the LQ survival curve fit as above.The Dose Modifying Factor (DMF) was determined by the quotient:

${{Dose}\mspace{14mu}{Modifying}\mspace{14mu}{Factor}\mspace{14mu}({DMF})} = \frac{D_{0}\left( {{Vehicle}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{D_{0}\left( {{Genistein}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}$

As a standard criterion, radiosensitization was defined as DMF>1.1.

The results of these experiments demonstrated that the nanoparticulategenistein composition had LET-dependent and cell line-specific effectson radiosensitivity of lung cancer cells. The genistein composition didnot change the radiosensitivity of H460 cells, regardless of LET orradiation type (FIGS. 2A and 2B), with a DMF of 1.07 for X-Rays and 1.04for higher LET protons. These cells showed modest, LET-dependentradiosensitization with a DMF of 1.12 for X-Rays vs higher LET protonsin the absence of genistein. In contrast, the nanoparticulate genisteincomposition exhibited clear LET-dependent radiosensitization for H1299cells, with a DMF of 0.97 for X-Rays and, interestingly, 1.35 for higherLET protons (FIGS. 3A and 3B). The LET (rather than radiation-type)specificity of this effect was confirmed in experiments using lower LETprotons in which the genistein composition failed to radiosensitizeH1299 cells (DMF=1.09). Finally, nanoparticulate genistein had nosignificant effect on clonogenic survival of normal human fibroblastcells, with a DMF of 1.01 and 1.07 for X-Rays and higher LET protons,respectively (FIGS. 4A and 4B). These results are summarized in TABLE 1.

TABLE 1 Cell survival after radiation exposure with and withoutgenistein pre-treatment X-Rays High LET Protons Cell Vehicle GenisteinVehicle Genistein Line (D₀) (D₀) DMF (D₀) (D₀) DMF H460 2.58 2.41 1.072.31 2.23 1.04 H1299 4.17 4.28 0.97 4.10 3.03 1.35 MRC5 1.40 1.38 1.011.35 1.26 1.07

In cancer radiotherapy, patients typically receive 60 Gy of radiation in2 Gy daily doses. A DMF of 1.35 means that patients who receive thenanoparticulate genistein composition disclosed herein would get a tumorequivalent of 60 Gy×1.35=81 Gy effective dose, such that the tumor wouldbe hit much harder by the radiation.

This information also can be used to determine the dose of high LETproton radiation necessary to effectively treat the tumor. Inparticular, a DMF of 1.35 means that rather than 60 Gy, a patienttreated with a nanoparticulate genistein composition could be treatedwith 60 Gy/1.35=44 Gy of high LET proton radiation to achieve the sametherapeutic effect as 60 Gy in a patient who did not receive thegenistein composition.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for sensitizing tumor cells to high linear energy transfer(LET) proton radiation, the method consisting of: contacting the tumorcells with a composition consisting essentially of genistein and one ormore pharmaceutically acceptable carriers, and subsequently contactingthe tumor cells with the high LET proton radiation, wherein thegenistein is effective to sensitize the tumor cells to the high LETproton radiation, and wherein the tumor cells are reduced in number andtherapeutically effectively treated with a dose of the high LET protonradiotherapy that is at least 10% less than a dose of high LET protonradiotherapy that would need to be administered to corresponding numberof tumor cells not treated with the genistein composition.
 2. The methodof claim 1, wherein the tumor cells are lung cancer cells, prostatecancer cells, head and neck cancer cells, pancreatic cancer cells,colon/colorectal cancer cells, bladder cancer cells, thyroid cancercells, breast cancer cells, liver cancer cells, ovarian cancer cells,endometrial cancer cells, cervical cancer cells, kidney cancer cells,brain cancer cells, or melanoma cells.
 3. The method of claim 1, whereinthe tumor cells are non-small cell lung cancer (NSCLC) cells.
 4. Themethod of claim 1, comprising contacting the tumor cells with thegenistein composition about 24 to 48 hours prior to contacting the tumorcells with the high LET proton radiation.
 5. A method for treating amammal identified as having a solid tumor and slated to undergotreatment with high LET proton radiotherapy, wherein the method consistsof: administering to the mammal a composition consisting essentially ofgenistein and one or more pharmaceutically acceptable carriers, andexposing the solid tumor in the mammal to high LET proton radiation,wherein the genistein is effective to sensitize cells in the tumor tothe high LET proton radiation, and wherein the tumor is therapeuticallyeffectively treated and the cells in the tumor are reduced in numberwith a dose of the high LET proton radiotherapy that is at least 10%less than a dose of high LET proton radiotherapy that would need to beadministered to a corresponding tumor in a mammal not treated with thecomposition.
 6. The method of claim 5, comprising administering thecomposition to the mammal at about 24 to 48 hours before the onset ofthe high LET proton radiotherapy.
 7. The method of claim 5, comprisingadministering the composition to the mammal at least once a day duringthe course of the high LET proton radiotherapy treatment.
 8. The methodof claim 5, wherein the tumor is a lung tumor, a prostate tumor, a headand neck tumor, a pancreatic tumor, a colon/colorectal tumor, a bladdertumor, a thyroid tumor, a breast tumor, a liver tumor, an ovarian tumor,an endometrial tumor, a cervical tumor, a kidney tumor, a brain tumor,or a melanoma.
 9. The method of claim 5, wherein the tumor is a NSCLCtumor.
 10. The method of claim 5, wherein the mammal is a human.
 11. Themethod of claim 5, comprising administering the composition to themammal at a dose of about 1,000 mg/day to 10,000 mg/day.
 12. The methodof claim 5, wherein the tumor comprises cells that express ERβ. 13-14.(canceled)
 15. The method of claim 5, wherein due to the administrationof the composition, the tumor is more effectively treated with the highLET proton radiotherapy, as compared to the effectiveness of the samedose of high LET proton radiotherapy administered to a correspondingtumor in a mammal not treated with the composition.
 16. The method ofclaim 5, wherein the one or more pharmaceutically acceptable carrierscomprise a water soluble polymer comprising a polyvinylpyrrolidone. 17.The method of claim 5, wherein the genistein is present in thecomposition at an amount ranging up to about 50% (w/w).
 18. (canceled)19. The method of claim 5, wherein the composition is formulated as atablet, a capsule, a powder, or a gelatin capsule.
 20. The method ofclaim 5, comprising administering the composition orally,intramuscularly, subcutaneously, or intravenously.
 21. The method ofclaim 1, wherein the cells are in a mammal.
 22. The method of claim 21,wherein a pharmaceutically acceptable sweetening agent is present in thecomposition.
 23. The method of claim 21, wherein the method comprisesadministering the composition to the mammal at a dose of about 1,000mg/day to 10,000 mg/day.
 24. The method of claim 5, wherein apharmaceutically acceptable sweetening agent is present in thecomposition.